The proposed research focuses on the determination of tertiary structures and internal dynamics of nucleic acids using electron paramagnetic resonance (EPR) spectroscopy. This incorporation of stable free radicals (spin labels) into the nucleic acids and data analysis of their EPR spectra. Specifically, we will simultaneously incorporate two spin-labels into complex nucleic acids, such as catalytic RNAs (ribozymes), and use spin-spin interactions to study the distances and relative orientation of helices within their tertiary structures. Furthermore, as EPR spectroscopy is an excellent probe of dynamics over a large range of motions we will study the dynamics of ribozymes under a variety of conditions that affect catalysis. These studies will provide information about the role of dynamics in ribozyme catalysis. The specific aims are: (1) To synthesize and characterize a new rigid spin label for incorporation into RNA. (2) To incorporate the new spin label into RNA duplexes and study duplex stability and structure. We will obtain a crystal structure of one spin labeled duplex in collaboration with Dr. Adrian Ferre-D'Amare. (3) To incorporate two spin labels into the structurally well characterized hammerhead ribozyme to dynamics of the hammerhead ribozyme will be determined under a variety of conditions that affect its catalytic activity. (4) To apply this approach for structural studies of the hairpin ribozyme. These studies will increase our understanding of ribozyme catalysis. Ths may lead to the design of improved RNA-catalysts, which are of therapeutic interest for antisense treatment of oncogenic and viral diseases. The proposed structural applications will be applicable to the determination of tertiary contacts between regions of non-catalytic RNAs and could be extended to the investigation RNA-protein interactions. Thus, the results of the proposed experiments will be of interest to those working in the areas of gene therapy, nucleic acids structure, protein-nucleic acid interactions, biomolecular catalysis and molecular recognition.